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Journal of Undergraduate Research

Keywords

phosphate homeostasis, transposon sequencing, E. coli, essential genes

College

Life Sciences

Department

Microbiology and Molecular Biology

Abstract

Though Escherichia Coli is a thoroughly investigated species of bacteria, questions still remain concerning genes involved in phosphate homeostasis. Phosphate homeostasis is the idea that a cell or bacterium adapts to changing environmental phosphate concentrations. Cells need to adapt to changing phosphate conditions because phosphate is essential to the biochemistry of many cellular processes. A cell therefore, must be able to collect and store phosphate when there is excess or scavenge and utilize stored phosphate in times when there are low levels of extracellular phosphate available. This process is not entirely understood in E.coli or other single celled organisms. A new technique has been developed in the last two years to analyze essential genes and their respective fitness levels. This technique is called Transposon Sequencing (Tn- Seq). Tn-Seq involves creating a library of bacteria that have transposons randomly inserted into their genome. The random insertions will block certain genes from being expressed. The library can then be challenged under certain conditions. While the library is challenged, only the bacteria without essential genes blocked by the transposons will grow under those challenge conditions. We can then take those bacteria, sequence them and see which non-essential genes were blocked by transposons, and which genes were untouched by transposons and therefore essential to the phosphate conditions. This experiment can be performed on large scale so we can see which “essential” genes affect the fitness by using a computer program to analyze the sequences. This large-scale analysis will help us develop an idea of how each “essential” gene affects the fitness of E.coli under the challenge conditions. We will employ this technique to determine which genes are essential for E.coli to survive in high, medium and low phosphate conditions. We can then analyze the amount of the bacteria that have each “essential” gene and to what amount and make conclusions about how each gene affects the fitness under those challenge conditions.

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